Project 1 (P1). Signaling mechanisms of toll-like and interleukin-1 receptors in inflammation Xiaoxia Li, Ph.D., Project Leader Project Summary/Abstract Obesity is a crucial public health problem that is associated with low-grade systemic inflammation and cardiovascular disease, affecting 25% of the population and its incidence is growing. IL-1R/TLRs have been directly implicated in obesity-associated inflammatory diseases. TLR2 or TLR4 deficiency reduced diet-induced insulin resistance and atherosclerosis, while human TLR4 null mutations are associated with reduced risk of atherosclerosis. The overall objective of this proposal is to elucidate IL-1R/TLR-mediated signaling mechanisms in the inflammatory response associated with metabolic diseases. Our long-term goals are to identify new therapeutic targets and to develop more effective anti-inflammatory small molecule drugs. Genetic and biochemical studies by us and others revealed that IL- 1R/TLRs interact with the adaptor MyD88 to initiate TAK1 (TGF?-activated kinase 1)- and MEKK3 (MAP kinase kinase kinase 3)-dependent pathways. Each pathway involves cascades of kinases organized by multiple adapter molecules into parallel and sequential signaling complexes, leading to activation of the transcription factor NF?B. IL- 1R/TLRs also mediate mRNA stabilization and promote protein translation of cytokines and chemokines through their impact on RNA binding proteins and translation mediators (mTOR and MnK1). We have previously shown that the kinase activity of IRAK4 (IL-1 receptor-associated kinase) is required for TAK1-dependent NF?B activation and posttranscriptional induction of cytokines and chemokines, but not for MEKK3-dependent NF?B activation. We have recently reported that while IRAK1 and IRAK2 interact with MyD88-IRAK4 to mediate TAK1-dependent NF?B activation and posttranscriptional control respectively; IRAKM (without functional kinase domain) mediates a second wave of IL-1R-TLR-induced NF?B activation through the MEKK3-dependent pathway, but also attenuates protein translation of cytokines and chemokines. In this proposal, we propose two Aims. In Aim 1, we will elucidate the molecular mechanism of IL-1R/TLRs signaling: (1) Delineate the precise molecular mechanism for IRAK1-mediated TAK1-dependent NF?B activation; (2) Investigate the mechanisms of IRAK2-mediated posttranscriptional control of cytokines and chemokines; (3) Elucidate the mechanisms by which IRAKM mediates MEKK3-dependent NF?B activation and its inhibitory effect on translational control of cytokines and chemokines. In Aim 2, we will investigate the mechanism of IL-1R/TLRs signaling for the development of obesity-associated diseases: (1) Study how IL- 1R/TLRs signaling mediate the cross-talk between adipocytes and macrophages contributing to diet-induced systemic inflammation and exacerbation on atherosclerosis; (2) Determine the impact of IL-1R/TLRs signaling in endothelial cells on the recruitment of monocytes and polarization of inflammatory macrophages.